Production of glycoproteins with reduced O-glycosylation

ABSTRACT

A method is described for producing protein compositions having reduced amounts of O-linked glycosylation. The method includes producing the protein in cells cultured in the presence of an inhibitor of Pmt-mediated O-linked glycosylation and/or in the presence of one or more α-1,2-mannosidases.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application claims benefit of International PatentApplication No. PCT/US2006/043535, which was filed 10 Nov. 2006, andU.S. Provisional Application No. 60/737,108, which was filed 15 Nov.2005.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to compositions and methods for producingproteins having specific glycosylation patterns. In particular, thepresent invention relates to compositions and methods for producingproteins having reduced O-linked glycosylation.

(2) Description of Related Art

Glycoproteins mediate many essential functions in humans and othermammals, including catalysis, signaling, cell-cell communication, andmolecular recognition and association. Glycoproteins make up themajority of non-cytosolic proteins in eukaryotic organisms (Lis andSharon, 1993, Eur. J. Biochem. 218:1-27). Many glycoproteins have beenexploited for therapeutic purposes, and during the last two decades,recombinant versions of naturally-occurring glycoproteins have been amajor part of the biotechnology industry. Examples of recombinantglycosylated proteins used as therapeutics include erythropoietin (EPO),therapeutic monoclonal antibodies (mAbs), tissue plasminogen activator(tPA), interferon-β(IFN-β), granulocyte-macrophage colony stimulatingfactor (GM-CSF), and human chorionic gonadotrophin (hCH) (Cumming etal., 1991, Glycobiology 1:115-130). Variations in glycosylation patternsof recombinantly produced glycoproteins have recently been the topic ofmuch attention in the scientific community as recombinant proteinsproduced as potential prophylactics and therapeutics approach theclinic.

In general, the glycosylation structures of glycoproteinoligosaccharides will vary depending upon the host species of the cellsused to produce them. Therapeutic proteins produced in non-human hostcells are likely to contain non-human glycosylation which may elicit animmunogenic response in humans—e.g. hypermannosylation in yeast (Ballou,1990, Methods Enzymol. 185:440-470); α(1,3)-fucose and β(1,2)-xylose inplants, (Cabanes-Macheteau et al., 1999. Glycobiology, 9: 365-372);N-glycolylneuraminic acid in Chinese hamster ovary cells (Noguchi etal., 1995. J. Biochem. 117: 5-62); and, Galα-1,3Gal glycosylation inmice (Borrebaeck, et al., 1993, Immun. Today, 14: 477-479). Carbohydratechains bound to proteins in animal cells include N-glycoside bond typecarbohydrate chains (also called N-glycans; or N-linked glycosylation)bound to an asparagine (Asn) residue in the protein and O-glycoside bondtype carbohydrate chains (also called O-glycans; or O-linkedglycosylation) bound to a serine (Ser) or threonine (Thr) residue in theprotein.

Because the oligosaccharide structures of glycoproteins produced bynon-human mammalian cells tend to be more closely related to those ofhuman glycoproteins, most commercial glycoproteins are produced inmammalian cells. However, mammalian cells have several importantdisadvantages as host cells for protein production. Besides beingcostly, processes for producing proteins in mammalian cells produceheterogeneous populations of glycoforms, have low volumetric titers, andrequire both ongoing viral containment and significant time to generatestable cell lines.

It is well recognized that the particular glycoforms on a protein canprofoundly affect the properties of the protein, including itspharmacokinetic, pharmacodynamic, receptor-interaction, andtissue-specific targeting properties (Graddis et al., 2002. Curr PharmBiotechnol. 3: 285-297). For example, it has been shown that differentglycosylation patterns of Igs are associated with different biologicalproperties (Jefferis and Lund, 1997, Antibody Eng. Chem. Immunol., 65:111-128; Wright and Morrison, 1997, Trends Biotechnol., 15: 26-32). Ithas further been shown that galactosylation of a glycoprotein can varywith cell culture conditions, which may render some glycoproteincompositions immunogenic depending on the specific galactose pattern onthe glycoprotein (Patel et al., 1992. Biochem J. 285: 839-845). However,because it is not known which specific glycoform(s) contribute(s) to adesired biological function, the ability to enrich for specificglycoforms on glycoproteins is highly desirable. Because differentglycoforms are associated with different biological properties, theability to enrich for glycoproteins having a specific glycoform can beused to elucidate the relationship between a specific glycoform and aspecific biological function of the glycoprotein. Also, the ability toenrich for glycoproteins having a specific glycoform enables theproduction of therapeutic glycoproteins having particular specificities.Thus, production of glycoprotein compositions that are enriched forparticular glycoforms is highly desirable.

While the pathway for N-linked glycosylation has been the subject ofmuch analysis, the process and function of O-linked glycosylation is notas well understood. However, it is known that in contrast to N-linkedglycosylation, O-glycosylation is a posttranslational event, whichoccurs in the cis-Golgi (Varki, 1993, Glycobiol., 3: 97-130). While aconsensus acceptor sequence for O-linked glycosylation like that forN-linked glycosylation does not appear to exist, a comparison of aminoacid sequences around a large number of O-linked glycosylation sites ofseveral glycoproteins show an increased frequency of proline residues atpositions −1 and +3 relative to the glycosylated residues and a markedincrease of serine, threonine, and alanine residues (Wilson et al.,1991, Biochem. J., 275: 529-534). Stretches of serine and threonineresidues in glycoproteins, may also be potential sites forO-glycosylation.

One gene family that has a role in O-linked glycosylation are the genesencoding the Dol-P-Man:Protein (Ser/Thr) Mannosyl Transferase (Pmt).These highly conserved genes have been identified in both highereukaryotes such as humans, rodents, insects, and the like and lowereukaryotes such as fungi and the like. Yeast such as Saccharomycescerevisiae and Pichia pastoris encode up to seven PMT genes encoding Pmthomologues (reviewed in Willer et al. Curr. Opin. Struct. Biol. 2003Oct.; 13(5): 621-30.). In yeast, O-linked glycosylation starts by theaddition of the initial mannose from dolichol-phosphate mannose to aserine or threonine residue of a nascent glycoprotein in the endoplasmicreticulum by one of the seven O-mannosyl transferases genes. While thereappear to be seven PMT genes encoding Pmt homologues in yeast,O-mannosylation of secreted fungal and heterologous proteins in yeast isprimarily dependent on the genes encoding Pmt1 and Pmt2, which appear tofunction as a heterodimer. PMT1 and PMT2 and their protein products,Pmt1 and Pmt2, respectively, appear to be highly conserved amongspecies.

Tanner et al. in U.S. Pat. No. 5,714,377 describes the PMT1 and PMT2genes of Saccharomyces cerevisiae and a method for making recombinantproteins having reduced O-linked glycosylation that uses fungal cells inwhich one or more of PMT genes have been genetically modified so thatrecombinant proteins are produced, which have reduced O-linkedglycosylation.

Ng et al. in U.S. Published Patent Application No. 20020068325 disclosesinhibition of O-glycosylation through the use of antisense orcosuppression or through the engineering of yeast host strains that haveloss of function mutations in genes associated with O-linkedglycosylation, in particular, one or more of the PMT genes.

UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyl-transferases (GalNAc-transferases) are involved in mucintype O-linked glycosylation found in higher eukaryotes. These enzymesinitiate O-glycosylation of specific serine and threonine amino acids inproteins by adding N-acetylgalactosamine to the hydroxy group of theseamino acids to which mannose residues can then be added in a step-wisemanner. Clausen et al. in U.S. Pat. No. 5,871,990 and U.S. PublishedPatent Application No. 20050026266 discloses a family of nucleic acidsencoding UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyl-transferases (GalNAc-transferases). Clausen in U.S.Published Patent Application No. 20030186850 discloses the use ofGalNAc-beta-benzyl to selectively inhibit lectins of polypeptideGalNAc-transferases and not serve as substrates for otherglycosyltransferases involved in O-glycan biosyntheses, thus inhibitingO-glycosylation.

Inhibitors of O-linked glycosylation have been described. For example,Orchard et al in U.S. Pat. No. 7,105,554 describes benzylidenethiazolidinediones and their use as antimycotic agents, e.g., antifungalagents. These benzylidene thiazolidinediones are reported to inhibit thePmt1 enzyme, preventing the formation of the O-linked mannoproteins andcompromising the integrity of the fungal cell wall. The end result iscell swelling and ultimately death through rupture.

Konrad et al. in U.S. Published Patent Application No. 20020128235disclose a method for treating or preventing diabetes mellitus bypharmacologically inhibiting O-linked protein glycosylation in a tissueor cell. The method relys on treating a diabetic individual with(Z)-1-[N-(3-Ammoniopropyl)-N-(n-propyl)amino] diazen-ium-1,2-diolate ora derivative thereof, which binds O-linked N-acetylglucosaminetransferase and thereby inhibits O-linked glycosylation.

Kojima et al. in U.S. Pat. No. 5,268,364 disclose therapeuticcompositions for inhibition of O-glycosylation using compounds such asbenzyle-α-N-acetylgalactosamine, which inhibits extension ofO-glycosylation leading to accumulation of O-α-GalNAc, to blockexpression of SLex or SLea by leukocytes or tumor cells and therebyinhibit adhesion of these cells to endothelial cells and platelets.

Boime et al. U.S. Pat. No. 6,103,501 disclose variants of hormones inwhich O-linked glycosylation was altered by modifying the amino acidsequence at the site of glycosylation.

The present inventors have found that particular chemical compounds thatare inhibitors of Pmt proteins, which are generally lethal to fungi, canbe used in a way which is not lethal to the host cells for production ofrecombinant proteins with reduced O-linked glycosylation. This enablesO-linked glycosylation of proteins produced from fungi and yeast cellsto be controlled. Other classes of chemical compounds, which theinventors believe to be non-lethal inhibitors of the PMT enzymes, arealso useful in the production of improved glycoproteins with reducedO-linked glycosylation. The present inventors have further found thataddition to the host cell or cell culture of certain classes of enzymes,namely, α-1,2-mannosidases, alone or in combination with a chemicalinhibitor of Pmt proteins effects a further reduction ofO-glycosylation.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods for producing proteins andglycoproteins having specific glycosylation patterns. In particular, thepresent invention provides a method for making a recombinant proteincompositions in a host cell in which the O-linked glycosylation of therecombinant protein is reduced by contacting the host cells with one ormore inhibitors of Pmt-mediated O-linked glycosylation of proteins inthe host cell or contacting the host cells or the recombinant proteinwith one or more α-1,2-mannosidases, or both. The amount of O-linkedglycosylation of the recombinant protein or glycoprotein is reducedcompared to the amount of O-linked glycosylation of the recombinantprotein or glycoprotein produced by the host cell in the absence of theinhibitor.

Pmt-mediated O-linked glycosylation refers to O-linked glycosylationwherein transfer of mannose residues to the serine or threonine residuesof a protein is mediated by a protein-O-D-mannosyltransferase (Pmt) orhomologue encoded by a PMT gene or its homologue. The inhibitors ofPmt-mediated O-linked glycosylation include inhibitors that inhibit anyone of the homologues of the PMT genes. In a currently preferred aspect,the inhibitor inhibits at least Pmt1 and/or Pmt2 activity of fungi andyeast, or the corresponding homologue in other organisms, including butnot limited to, mammals, plants, and insects.

Currently, it is preferable that the amount of O-linked glycosylationhas been reduced through the use of a chemical inhibitor, for example, achemical inhibitor encompassed by the class of chemicals calledbenzylidene thiazolidinediones. In particular embodiments, the chemicalinhibitor is selected from the group consisting of5-[[3,4-bis(phenylmethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticacid;5-[[3-(1-Phenylethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticacid; 3-Hydroxy-4-(2-phenylethoxy)benzaldehyde;3-(1-Phenylethoxy)-4-(2-phenylethoxy)-benzaldehyde;5-[[3-(1-Phenyl-2-hydroxy)ethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticacid.

In further aspects, provided is a method for producing recombinantprotein compositions having reduced O-linked glycosylation, which useone or more inhibitors of the Pmt proteins involved in O-linkedglycosylation and/or one or more α-1,2-mannosidase enzymes to producethe protein having reduced O-linked glycosylation. Currently preferredα-1,2-mannosidases may be isolated from eukaryotic cells, includingmammalian and yeast cells. In currently preferred embodiments, theα-1,2-mannosidase is that produced by Trichoderma reesei, Saccharomycessp., or Aspergillus sp. In other currently preferred embodiments, theα-1,2-mannosidase may be produced from a chimeric construct comprising anucleic acid sequence encoding the catalytic domain of anα-1,2-mannosidase operatively linked to a nucleic acid sequence encodinga cellular targeting signal peptide not normally associated with thecatalytic domain. In other embodiments, the α-1,2-mannosidase may beseparately produced and added to the cell culture, or may be produced byco-expressing the α-1,2-mannosidase with the recombinant glycoprotein.

In particular aspects of the method, the recombinant protein compositioncomprises a glycoprotein having N-linked glycosylation wherein therecombinant glycoprotein includes at least one predominant N-glycoformand has reduced O-linked glycosylation. Therefore, further provided areglycoprotein compositions comprising a predominant species of N-glycanstructure and having reduced O-linked glycosylation compared tocompositions of the glycoprotein which have been produced in host cellshave not been incubated in the presence of an inhibitor of Pmt-mediatedO-linked glycosylation or an α-1,2-mannosidase capable of trimming morethan one mannose residue from a glycans structure. In particularaspects, the glycoprotein composition comprises a glycoprotein having apredominant N-glycan structure selected from the group consisting ofMan₅GlcNAc₂, Man₃GlcNAc₂, GlcNAcMan₅GlcNAc₂, GlcNAcMan₃GlcNAc₂,GlcNAc₂Man₃GlcNAc₂, GalGlcNAcMan₅GlcNAc₂, Gal(GlcNAc)₂ Man₅GlcNAc₂,(GalGlcNAc)₂Man₅GlcNAc₂, NANAGalGlcNAcMan₃GlcNAc₂,NANA₂Gal₂GlcNAcMan₃GlcNAc₂, and GalGlcNAcMan₃GlcNAc₂ glycoforms. Animportant aspect of the method is that it provides for a glycoproteincomposition comprising reduced O-linked glycosylation and predominantlya specific N-linked glycoform in which the recombinant glycoprotein mayexhibit increased biological activity and/or decreased undesiredimmunogenicity relative to compositions of the same glycoproteinproduced from mammalian cell culture, such as CHO cells. An additionaladvantage of producing the glycoprotein composition comprising reducedO-linked glycosylation and a predominant N-linked glycoform is that itavoids production of undesired or inactive glycoforms and heterogeneousmixtures, which may induce undesired effects and/or dilute the moreeffective glycoform. Thus, therapeutic pharmaceutical composition ofglycoprotein molecules comprising, for example, predominantlyMan₅GlcNAc₂, Man₃GlcNAc₂, GlcNAcMan₅GlcNAc₂, GlcNAcMan₃GlcNAc₂,GlcNAc₂Man₃ GlcNAc₂, GalGlcNAcMan₅GlcNAc₂, Gal(GlcNAc)₂ Man₅GlcNAc₂,(GalGlcNAc)₂Man₅GlcNAc₂, NANAGalGlcNAcMan₃GlcNAc₂,NANA₂Gal₂GlcNAcMan₃GlcNAc₂, and GalGlcNAcMan₃GlcNAc₂ glycoforms andhaving reduced O-linked glycosylation may well be effective at lowerdoses, thus having higher efficacy/potency.

Therefore, provided is a method of producing a protein having reducedO-linked glycosylation comprising providing a nucleic acid encoding aprotein; introducing the nucleic acid into a host cell to provide aculture of the host cell; contacting the culture with one or moreinhibitors of Pmt-mediated O-linked glycosylation; and isolating theglycoprotein produced by the host cell in the presence of the inhibitorto produce the protein having reduced O-linked glycosylation.

In particular aspects of the method, the culture is grown for a timesufficient to provide a multiplicity of the host cells having thenucleic acid before contacting the culture with the one or moreinhibitors of Pmt-mediated O-linked glycosylation or the culture isgrown in the presence of the one or more inhibitors of Pmt-mediatedO-linked glycosylation at the time the culture is established.

In a further aspect of the method, the nucleic acid encoding the proteinis operably linked to an inducible promoter. Then the culture is grownfor a time sufficient to provide a multiplicity of the host cells havingthe nucleic acid before contacting the culture with the one or moreinhibitors of Pmt-mediated O-linked glycosylation and an inducer of thepromoter to induce expression of the protein and isolating the proteinproduced by the host cell in the presence of the one or more inhibitorsand the inducer to produce the protein having reduced O-linkedglycosylation or the culture is contacted with an inducer of thepromoter to induce expression of the protein for a time beforecontacting the culture with the one or more inhibitors of Pmt-mediatedO-linked glycosylation and isolating the protein produced by the hostcell in the presence of the inhibitor and the inducer to produce theprotein having reduced O-linked glycosylation.

Further provided is a method of producing a protein having reducedO-inked glycosylation comprising providing a nucleic acid encoding aprotein; introducing the nucleic acid into a host cell to provide aculture of the host cell; contacting the culture with one or moreα-1,2-mannosidase enzymes; and isolating the protein produced by thehost cell in the presence of the one or more α-1,2-mannosidase enzymesto produce the glycoprotein having reduced O-linked glycosylation.

In particular aspects of the method, the culture is grown for a timesufficient to provide a multiplicity of the host cells having thenucleic acid before contacting the culture with the one or moreα-1,2-mannosidase enzymes cosylation. In other aspects, the culture isgrown in the presence of the one or more α-1,2-mannosidase enzymes.

In further aspects of the method, a second nucleic acid encoding the oneor more α-1,2-mannosidase enzymes is provided and introducing the secondnucleic acid into the host cell. In particular aspects, a second nucleicacid encoding the one or more α-1,2-mannosidase enzymes operably linkedto an inducible promoter is provided and introducing the second nucleicacid into the host cell and the culture is grown for a time sufficientto provide a multiplicity of the host cells before inducing expressionof the protein and the one or more α-1,2-mannosidase enzymes to producethe protein having reduced O-linked glycosylation or expression of theprotein is induced for a time before inducing expression of the one ormore α-1,2-mannosidase enzymes to produce the protein having reducedO-linked glycosylation or expression of the one or moreα-1,2-mannosidase enzymes is induced for a time before inducingexpression of the protein to produce the protein having reduced O-linkedglycosylation.

Further provided is a method of producing a protein having reducedO-linked glycosylation comprising providing a nucleic acid encoding aprotein operably linked to an inducible promoter; introducing thenucleic acid into a host cell and growing the host cell containing thenucleic acid to produce a culture of the host cell; contacting theculture with one or more inhibitors of Pmt-mediated O-linkedglycosylation and one or more one or more α-1,2-mannosidase enzymes; andisolating the glycoprotein produced by the host cell in the presence ofthe one or more inhibitors and the one or more one or moreα-1,2-mannosidase enzymes to produce the protein having reduced O-linkedglycosylation.

In particular aspects of the method, the culture is grown for a timesufficient to provide a multiplicity of the host cells having thenucleic acid before contacting the culture with the one or moreinhibitors of Pmt-mediated O-linked glycosylation or the culture isgrown in the presence of the one or more inhibitors of Pmt-mediatedO-linked glycosylation at the time the culture is established.

In a further aspect of the method, the nucleic acid encoding the proteinis operably linked to an inducible promoter. Then the culture is grownfor a time sufficient to provide a multiplicity of the host cells havingthe nucleic acid before contacting the culture with the one or moreinhibitors of Pmt-mediated O-linked glycosylation and an inducer of thepromoter to induce expression of the protein and isolating the proteinproduced by the host cell in the presence of the one or more inhibitorsand the inducer to produce the protein having reduced O-linkedglycosylation or the culture is contacted with an inducer of thepromoter to induce expression of the protein for a time beforecontacting the culture with the one or more inhibitors of Pmt-mediatedO-linked glycosylation and isolating the protein produced by the hostcell in the presence of the inhibitor and the inducer to produce theprotein having reduced O-linked glycosylation.

In particular aspects of the method, the culture is grown for a timesufficient to provide a multiplicity of the host cells having thenucleic acid before contacting the culture with the one or moreα-1,2-mannosidase enzymes. In other aspects, the culture is grown in thepresence of the one or more α-1,2-mannosidase enzymes.

In further aspects of the method, a second nucleic acid encoding the oneor more α-1,2-mannosidase enzymes is provided and introducing the secondnucleic acid into the host cell. In particular aspects, a second nucleicacid encoding the one or more α-1,2-mannosidase enzymes operably linkedto an inducible promoter is provided and introducing the second nucleicacid into the host cell and the culture is grown for a time sufficientto provide a multiplicity of the host cells before inducing expressionof the protein and the one or more α-1,2-mannosidase enzymes to producethe protein having reduced O-linked glycosylation or expression of theprotein is induced for a time before inducing expression of the one ormore α-1,2-mannosidase enzymes to produce the protein having reducedO-linked glycosylation or expression of the one or moreα-1,2-mannosidase enzymes is induced for a time before inducingexpression of the protein to produce the protein having reduced O-linkedglycosylation.

In further aspects of the above methods that use one or more inhibitorsof a Pmt protein, currently, it is preferred that the one or moreinhibitors is selected from the class of molecules comprisingbenzylidene thiazolidinediones. Currently, it is preferable that the oneor more inhibitors be selected from the group consisting of5-[[3,4-bis(phenylmethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticAcid;5-[[3-(1-Phenylethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticAcid; and5-[[3-(1-Phenyl-2-hydroxy)ethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticAcid.

In particular aspects of the above methods that use anα-1,2-mannosidase, it is currently preferable that the α-1,2-mannosidaseis selected from the group consisting of Trichoderma reesei,Saccharomyces sp., and Aspergillus sp. Currently, it is preferable thatthe α-1,2-mannosidase is from Trichoderma reesei. Alternatively, thehost cell can include in addition to the first nucleic acid encoding theprotein or glycoprotein, a second nucleic acid, which encodes theα-1,2-mannosidase, operably linked to an inducible promoter. Expressionof the α-1,2-mannosidase and the protein or glycoprotein can be inducedsimultaneously or expression of the protein or glycoprotein inducedbefore expression of the α-1,2-mannosidase or vice versa.

While the method can be performed using any host cell that producedproteins having O-linked glycosylation, in currently preferred aspects,the host cell is a lower eukaryotic cell, preferably a fungal cell or ayeast cell. Currently, it is preferred that the host cell be selectedfrom the group consisting of cells from K. lactis, Pichia pastoris,Pichia methanolica, and Hansenula. In further embodiments for producingrecombinant glycoproteins in particular, the host cell is a yeast orfilamentous fungal cell that has been genetically modified to produceglycoproteins with predominantly a particular N-glycan structure. Inparticularly preferred aspects, the host cells are genetically modifiedso that they express recombinant glycoproteins in which theglycosylation pattern is human-like or humanized. In particular, thehost cells can be modified so that they express recombinantglycoproteins having predominantly a particular desired N-glycanstructure. A lower eukaryotic host cell when used herein in connectionwith glycosylation profiles, refers to any eukaryotic cell whichordinarily produces high mannose containing N-linked glycans, and thus,includes most typical lower eukaryotic cells, including uni- andmulti-cellular fungal and algal cells.

All publications, patents, patent applications, and other referencesmentioned herein are hereby incorporated by reference in theirentireties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the effect of Pmt inhibitors on O-glycosylation ofsecreted recombinant reporter proteins in Pichia pastoris. The chemicalinhibitors of Pmt reduced O-glycosylation to a level similar to thatobserved in a strain lacking PMT1. Western blotting using ananti-polyhistidine antibody was used to detect His-tagged human Kringle1-3 domain (K1-3) of human plasminogen in the growth media of wild-type(lanes 1-3) and pmt1 (lanes 4-5) strains. The slower migrating bands(seen as a higher molecular weight smear for K1-3 in lane 1) indicateO-glycosylated protein. Pmti-1, PMT inhibitor 1.

FIG. 2 shows a Western blot that demonstrates the effect of T. reeseiα-mannosidase and the chemical inhibitor Pmti-2 on the O-glycosylationof immunoglobulin light and heavy chain polypeptides. Both T. reeseiα-mannosidase and chemical Pmt inhibitors reduced the level ofO-glycosylation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for expressing a recombinantprotein (includes polypeptides and glycoproteins), which is susceptibleto O-linked glycosylation in a particular host cell, having a reducedamount of O-linked glycosylation (including no O-linked glycosylation)in that cell type. The method involves inducing expression of a proteinof interest in a host cell in which the protein is susceptible toO-linked glycosylation in the host cell in the presence of a chemicalinhibitor of the activity of one or more of the Dol-P-Man:Protein(Ser/Thr) Mannosyl Transferase (Pmt) proteins involved in the transferof mannose to a serine or threonine residue of the protein in the cellor one or more α 1,2-mannosidases, or both, at the time expression ofthe protein is induced. The protein that is expressed in the presence ofthe inhibitor or the one or more α 1,2-mannosidases has a reduced amountof O-linked glycosylation compared to the amount of O-linkedglycosylation that would have been present on the protein if it had beenproduced in the absence of the inhibitor or the one or more α1,2-mannosidases, or both. The method is particularly useful because itprovides a means for producing therapeutically relevant proteins whereit is desired that the protein have a reduced amount of O-glycosylationin host cells such as lower eukaryotes, for example yeast, and bacteria,which would normally produce proteins with O-linked glycans, having areduced number of O-linked glycans. However, while the method isespecially suitable for expressing proteins with reduced O-linkedglycosylation in lower eukaryotic organisms, the method can also bepracticed in higher eukaryotic organisms and bacteria.

The method is an improvement over prior art methods for producingproteins having reduced O-linked glycosylation in host cells in whichthe proteins are susceptible to O-linked glycosylation. For example,Tanner et al. in U.S. Pat. No. 5,714,377 describes a method for makingrecombinant proteins having reduced O-linked glycosylation using fungalcells such as yeast cells in which one or more of PMT genes encoding thePmt protein have been genetically modified so that recombinant proteinsare produced, which have reduced O-linked glycosylation. While deletionof either the PMT1 or PMT2 genes in a fungal host cell enablesproduction of a recombinant protein having reduced O-linkedglycosylation in the fungal host cell, expression of the PMT1 and PMT2genes are important for host cells growth and either deletion alone alsoadversely affects the ability of the fungal host cell to grow thusmaking it difficult to produce a sufficient quantity of host cells orrecombinant protein with a reduced amount of O-linked glycosylation.Deletion of both genes appears to be lethal to the fungal host cell.Therefore, genetic elimination of the PMT1 and PMT2 genes in a host cellwould appear to be an undesirable means for producing recombinantproteins having reduced O-linked glycosylation.

In contrast, the PMT genes in the host cells used in the method of thepresent invention have not been modified or deleted, which enables thehost cell to O-glycosylate those proteins that are important for cellgrowth until which time the activity of the Pmt proteins is inhibited.In general, this enables the host cells to be grown to higher levelsthan the levels that could be obtained if the PMT genes had beendeleted. In addition, in particular embodiments, expression of therecombinant protein in the host cell is controlled by an induciblepromoter and the Pmt activity in the host cell is not inhibited or oneor more α 1,2-mannosidases added, or both, until expression of therecombinant protein is induced. This enables large quantities of hostcells containing a nucleic acid encoding a recombinant protein to beproduced in culture before inducing expression of the recombinantprotein and adding the Pmt inhibitor and/or one or more α1,2-mannosidases. This can enable production of larger amountsrecombinant protein having reduced O-linked glycosylation to be producedin the culture in a shorter period of time than would occur for hostcells which have had one or more PMT genes deleted and grow poorly inculture.

This improvement over the prior art also facilitates the production ofglycoproteins having reduced O-linked glycosylation in host cells thathave been genetically modified to produce glycoproteins havingpredominantly a particular N-linked glycan structure but which alsoO-glycosylate the glycoprotein. Methods for producing a wide variety ofglycoproteins having predominantly particular N-linked glycoforms havebeen disclosed in U.S. Pat. No. 7,029,872 and U.S. Published ApplicationNos. 20050170452, 20050260729, 20040230042, 20050208617, 20050208617,20040171826, 20060160179, 20060040353, and 20060211085. Any one of thehost cells described in the aforementioned patent and patentapplications can be used to produce a glycoprotein having predominantlya particular N-linked glycan structure and having reduced O-linkedglycosylation using the method disclosed herein. It has been found thatsome host cells that have been genetically modified to produceglycoproteins having predominantly a particular N-linked glycanstructure can grow less well in culture under particular conditions thanhost cells that have not been modified. For example, particular fungaland yeast cells in which genes involved in hypermannosylation have beendeleted and other genes needed to produce particular mammalian or humanlike N-linked glycan structures have been added, can grow less well thanfungal or yeast cells that do not the genetic modifications. In some ofthese genetically modified fungal or yeast cells, further introducingdeletions of the PMT1 or PMT2 genes either is lethal to the cells oradversely affects the ability of the cells to grow to sufficientquantities in culture. The method herein avoids the potentialdeleterious effects of deleting the PMT1 and PMT2 genes by allowing thecells to grow to sufficient quantities in culture before inducingexpression of the recombinant glycoprotein and adding an inhibitor ofthe activity of the Pmt proteins, or one or more α 1,2-mannosidases, orboth, to produce the recombinant glycoprotein having predominantlyparticular N-linked glycan structures and reduced O-linkedglycosylation.

Therefore, an important aspect of the method is that it provides for aglycoprotein composition comprising reduced O-linked glycosylation and apredominantly a specific N-linked glycoform in which the recombinantglycoprotein may exhibit increased biological activity and/or decreasedundesired immunogenicity relative to compositions of the sameglycoprotein produced from mammalian cell culture, such as CHO cells. Anadditional advantage of producing the glycoprotein compositioncomprising reduced O-linked glycosylation and a predominant N-linkedglycoform is that it avoids production of undesired or inactiveglycoforms and heterogeneous mixtures, which may induce undesiredeffects and/or dilute the more effective glycoform. Thus, therapeuticpharmaceutical composition of glycoprotein molecules comprising, forexample, predominantly Man₅GlcNAc₂, Man₃ GlcNAc₂, GlcNAcMan₅GlcNAc₂,GlcNAcMan₃ GlcNAc₂, GlcNAc₂Man₃GlcNAc₂, GalGlcNAcMan₅GlcNAc₂,Gal(GlcNAc)₂ Man₅GlcNAc₂, (GalGlcNAc)₂Man₅GlcNAc₂,NANAGalGlcNAcMan₃GlcNAc₂, NANA₂Gal₂GlcNAcMan₃GlcNAc₂, andGalGlcNAcMan₃GlcNAc₂ glycoforms and having reduced O-linkedglycosylation may well be effective at lower doses, thus having higherefficacy/potency.

In general, the method for producing proteins having reduced O-linkedglycosylation comprises transforming a host cell with a nucleic acidencoding a recombinant or heterologous protein in which it is desirableto produce the protein having reduced O-linked glycosylation. Thenucleic acid encoding the recombinant protein is operably linked toregulatory sequences that allow expression of the recombinant protein.Such regulatory sequences include an inducible promoter and optionallyan enhancer upstream, or 5′, to the nucleic acid encoding the fusionprotein and a transcription termination site 3′ or down stream from thenucleic acid encoding the recombinant protein. The nucleic acid alsotypically encodes a 5′ UTR region having a ribosome binding site and a3′ untranslated region. The nucleic acid is often a component of avector replicable in cells in which the recombinant protein isexpressed. The vector can also contain a marker to allow recognition oftransformed cells. However, some cell types, particularly yeast, can besuccessfully transformed with a nucleic acid lacking extraneous vectorsequences.

Nucleic acids encoding desired recombinant proteins can be obtained fromseveral sources. cDNA sequences can be amplified from cell lines knownto express the protein using primers to conserved regions (see, forexample, Marks et al., J. Mol. Biol. 581-596 (1991)). Nucleic acids canalso be synthesized de novo based on sequences in the scientificliterature. Nucleic acids can also be synthesized by extension ofoverlapping oligonucleotides spanning a desired sequence (see, e.g.,Caldas et al., Protein Engineering, 13, 353-360 (2000)).

In one aspect, the nucleic acid encoding the protein is operably linkedto an inducible promoter, which allows expression of the protein to beinduced when desired. In another aspect, the nucleic acid encoding theprotein is operably linked to a constitutive promoter. To facilitateisolation of the expressed protein, it is currently preferable that theprotein include a signal sequence that directs the protein to beexcreted into the cell culture medium where it can then be isolated. Inthe first aspect, the transformed host cells are cultured for a timesufficient to produce a desired multiplicity of host cells sufficient toproduce the desired amount of protein before adding one or moreinhibitors of Pmt-mediated O-linked glycosylation to the culture medium.The inducer and inhibitor can be added to the culture simultaneously orthe inducer is added to the culture before adding the one or more Pmtinhibitors or the one or more Pmt inhibitors is added to the culturebefore adding the inducer. The induced protein is produced havingreduced O-linked glycosylation and can be recovered from the culturemedium or for proteins not having a signal sequence, from the host cellby lysis. In the second aspect, wherein the nucleic acid encoding theprotein is operably linked to a constitutive promoter, the one or moreinhibitors of Pmt-mediated O-linked glycosylation is added to theculture medium at the same time the culture is established and theprotein, which is produced having reduced O-linked glycosylation, can berecovered from the culture medium or for proteins not having a signalsequence, from the host cell by lysis. An example illustrating themethod using an inducible promoter is shown in Example 2 and an exampleillustrating the method using a constitutive promoter is shown inExample 3.

Inhibitors useful for producing proteins with reduced O-linkedglycosylation are chemicals or compositions that inhibit the activityone or more of the Pmt proteins. When the host cell is a lower eukaryotesuch as fungi or yeast, it is desirable that the inhibitor inhibit atleast the activity of Pmt1 or Pmt2, or both. In higher eukaryotes, it isdesirable that the inhibitor inhibit activity of the homologue in thehigher eukaryote that corresponds to the Pmt1 or Pmt2. Chemicalinhibitors that can be used include the benzylidene thiazolidinedionesidentified in U.S. Pat. No. 7,105,554, which includes5-[[3,4-bis(phenylmethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticacid;5-[[3-(1-phenylethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticacid; 3-hydroxy-4-(2-phenylethoxy)benzaldehyde;3-(1-phenylethoxy)-4-(2-phenylethoxy)-benzaldehyde; and,5-[[3-(1-phenyl-2-hydroxy)ethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticacid. Other compounds that might be useful are the structurally similarcompounds disclosed in Voss et al. in WO 94/29287, which disclosesmethods of making arylidene-4-oxo-2-thioxo-3-thiazolidine carboxylicacids and which are disclosed to be useful in the prophylaxis andtreatment of late effects of diabetes as well as the prophylaxis andtreatment of atherosclerosis and arteriosclerosis and in Esswein et al.in U.S. Pat. No. 6,673,816, which discloses methods of makingderivatives of rhodaninecarboxylic acids and their use for treatment ofmetabolic bone disorders.

In the examples, chemical inhibitors selected from the group consistingof5-[[3,4-bis(phenylmethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticacid;5-[[3-(1-phenylethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticacid; and,5-[[3-(1-phenyl-2-hydroxy)ethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticacid are shown to be effective in producing recombinant proteins havingreduced O-linked glycosylation in Pichia pastoris strains that hadintact, functional PMT1 and PMT2 genes. Table 1 of Example 2 shows thatany one of the above three Pmt chemical inhibitors added to a culture ofrecombinant Pichia pastoris having intact, functional PMT1 and PMT2genes and transformed with a nucleic acid encoding a recombinant,secretable Kringle 1-3 protein operably linked to an inducible promoterat the time expression of the recombinant protein was induced, produceda recombinant protein having a level of reduced O-linked glycosylationthat was comparable to the level of O-linked glycosylation seen forPichia pastoris cells containing a deletion of either the PMT1 or PMT2gene. The above Pmti inhibitors have been used in amounts from about0.03 μM to 20 μM to produce proteins having reduced O-linkedglycosylation compared to the amount of O-linked glycosylation on theprotein when grown in similar host cell cultures in the absence of thePmti inhibitors. The results shown in Example 3 further shows that thehost cell cultures can be grown in the presence of Pmti inhibitor at anamount that is sufficient to inhibit O-linked glycosylation withoutkilling the host cells.

The method can include adding to the culture medium containing the oneor more Pmt inhibitors one or more α-1,2-mannosidase enzymes to producethe recombinant protein having reduced O-linked glycosylation. Theα-1,2-mannosidases are a conserved family of eukaryotic enzymes formaturation of N-glycans, which are capable of trimming Man₉GlcNAc₂ toMan₈GlcNAc₂ in yeast. (Vallee et al., 2000, EMBO J., 19: 581-588). Theα-1,2-mannosidases are also known as class I α-mannosidases and havebeen identified in mammalian, lower eukaryotic species, and insect cells(Kawar et al., 2000, Glycobiology 10: 347-355). Mammalian cells areknown to have several class I α-mannosidases, some of which are capableof trimming multiple mannose residues (Moremen et al., 1994,Glycobiology 4: 113-125), while yeast appear to have fewer, morespecialized α-1,2-mannosidases. For example, Saccharomyces has beendisclosed to have a single α-1,2-mannosidase encoded by MNN1, whichremoves one specific mannose residue (for example, Man₉GlcNAc₂ toMan₈GlcNAc₂) (Herscovics, 1999, Biochim Biophys Acta., 1473: 96-107).Thus, the endogenous α-1,2-mannosidase present in many lower eukaryotessuch as fungi and yeast and which cannot remove multiple mannoseresidues from Glycan structures, is not capable of enabling productionof proteins having reduced O-linked glycosylation. Therefore, the methodherein requires introduction into the culture medium containing the hostcells an α-1,2-mannosidase capable of trimming multiple mannose residuesfrom an O-linked Glycan or introduction into the host cell a nucleicacid encoding an α-1,2-mannosidase capable of trimming multiple mannoseresidues from an O-linked Glycan. The α-1,2-mannosidase herein includesthe intact, native α-1,2-mannosidase; an α-1,2-mannosidase modified toenhance its α-1,2-mannosidase activity; an α-1,2-mannosidase modified todecrease its α-1,2-mannosidase activity; and, a recombinantα-1,2-mannosidase comprising at least the catalytic domain having theα-1,2-mannosidase activity (for example, a fusion protein comprising thecatalytic domain having the α-1,2-mannosidase activity fused toheterologous proteins, polypeptides, or peptides).

In particular embodiments, the α-1,2-mannosidase, which is capable oftrimming multiple mannose residues from an O-linked glycans and is addedto the cell culture, is produced by Trichoderma sp., Saccharomyces sp.,or Aspergillus sp. Currently, preferred α-1,2-mannosidases are obtainedfrom Trichoderma reesei, Aspergillus niger, or Aspergillus oryzae. T.reesei is also known as Hypocrea jecorina. In Example 3, a transformedyeast comprising an expression cassette, which expresses a recombinantα-1,2-mannosidases comprising the Trichoderma reesei α-1,2-mannosidasecatalytic domain fused to the Saccharomyces cerevisiea αMAT pre signalsequence, was used to produce recombinant proteins having reducedO-linked glycosylation. Another example of a recombinantα-1,2-mannosidase that could be used in the method herein to produceproteins having reduced O-linked glycosylation is the recombinantTrichoderma reesei α-1,2-mannosidase disclosed in Maras et al., 2000, J.Biotechnol. 77:255-263 wherein the Trichoderma reesei α-1,2-mannosidasecatalytic domain was fused to a Saccharomyces cerevisiea α-MATprepro-signal peptide.

The α-1,2-mannosidase can also be produced from a chimeric nucleic acidcomprising a nucleic acid sequence encoding at least the catalyticdomain of an α-1,2-mannosidase, which is capable of trimming multiplemannose residues from an O-linked glycans, operatively linked to anucleic acid sequence encoding a cellular targeting signal peptide notnormally associated with the catalytic domain. The chimeric nucleic acidcan be operably linked to a constitutive or inducible promoter. Thechimeric nucleic acid is transformed into a host cell to produce theα-1,2-mannosidase, which is then isolated and then added to the cellculture medium containing cells transformed with the nucleic acidencoding the heterologous protein at the time expression of the proteinis induced. Alternatively, the host cell is transformed with thechimeric nucleic acid encoding the α-1,2-mannosidase and the nucleicacid encoding the recombinant protein and co-expressing theα-1,2-mannosidase and the recombinant protein at the same time. Inparticular embodiments, both the chimeric nucleic acid encoding theα-1,2-mannosidase and the nucleic acid encoding the recombinant proteinas both operably linked to an inducible promoter. In other embodiments,one or both of the promoters are constitutive. Example 3 illustrates themethod wherein nucleic acids encoding both the α-mannosidase and therecombinant protein are operably linked to a constitutive promoter,introduced into a host cell, and a culture of the host cells is thenincubated in the presence of one or more Pmt inhibitors to produce therecombinant protein having reduced O-linked glycosylation. Example 3further shows that there appears that the Pmti inhibitor and theα-1,2-mannosidase appear to synergistically reduce the amount ofO-linked glycosylation compared to the amount of O-linked glycosylationin the presence of either alone.

In particular aspects, reduced O-linked glycosylation can be effected byadding only the one or more α-1,2-mannosidases and not one or more Pmtinhibitors to the culture medium. In one aspect, the nucleic acidencoding the recombinant protein is operably linked to an induciblepromoter, which allows expression of the recombinant protein to beinduced when desired. In another aspect, the nucleic acid encoding theprotein is operably linked to a constitutive promoter. To facilitateisolation of the expressed recombinant protein, it is currentlypreferable that the protein include a signal sequence that directs therecombinant protein to be excreted into the cell culture medium where itcan then be isolated.

In the first aspect, the transformed host cells are cultured for a timesufficient to produce a desired multiplicity of host cells sufficient toproduce the desired amount of the recombinant protein before adding theone or more α-1,2-mannosidases to the culture medium. The inducer andthe one or more α-1,2-mannosidases can be added to the culturesimultaneously or the inducer is added to the culture before adding theone or more α-1,2-mannosidases or the one or more α-1,2-mannosidases isadded to the culture before adding the inducer. The induced recombinantprotein is produced having reduced O-linked glycosylation and can berecovered from the culture medium or for proteins not having a signalsequence, from the host cell by lysis.

In the second aspect, wherein the nucleic acid encoding the recombinantprotein is operably linked to a constitutive promoter, the one or moreα-1,2-mannosidases is added to the culture medium at the same time theculture is established and the recombinant protein, which is producedhaving reduced O-linked glycosylation, can be recovered from the culturemedium or for recombinant proteins not having a signal sequence, fromthe host cell by lysis.

In a further still aspect for producing proteins having reduced O-linkedglycosylation without using an inhibitor of Pmt-mediated O-linkedglycosylation, the host cell is transformed with a chimeric nucleic acidencoding the α-1,2-mannosidase and a nucleic acid encoding therecombinant protein and co-expressing the α-1,2-mannosidase and therecombinant protein to produce the recombinant protein having reducedO-linked glycosylation. In particular embodiments, both the chimericnucleic acid encoding the α-1,2-mannosidase and the nucleic acidencoding the recombinant protein as both operably linked to an induciblepromoter. In other embodiments, one or both of the promoters areconstitutive. In the case of an inducible promoter, the host cells aregrown to produce a desired multiplicity of host cells before inducingexpression of the α-1,2-mannosidase and/or recombinant protein. Example3 illustrates the method wherein nucleic acids encoding both theα-1,2-mannosidases and the recombinant protein are operably linked to aconstitutive promoter are introduced into a host cell and a culture ofthe host cells is then incubated for a time to produce the recombinantprotein, which has reduced O-linked glycosylation compared to therecombinant protein produced in cells in the absence of theα-1,2-mannosidase.

II. Host Cells

While host cells for the method herein includes both higher eukaryotecells and lower eukaryote cells, lower eukaryote cells, for examplefilamentous fungi or yeast cells, are currently preferred for expressionof proteins because they can be economically cultured, give high yieldsof protein, and when appropriately modified are capable of producingproteins having suitable glycosylation patterns. Lower eukaryotesinclude yeast, fungi, collar-flagellates, microsporidia, alveolates(e.g., dinoflagellates), stramenopiles (e.g, brown algae, protozoa),rhodophyta (e.g., red algae), plants (e.g., green algae, plant cells,moss) and other protists. Yeast and fungi include, but are not limitedto: Pichia sp. (for example, Pichia pastoris, Pichia finlandica, Pichiatrehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta(Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichiathermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi,Pichia stiptis, Pichia methanolica), Saccharomyces sp. (for exampleSaccharomyces cerevisiea), Hansenula polymorpha, Kluyveromyces sp. (forexample, Kluyveromyces lactis), Candida albicans, Aspergillus sp (forexample, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae),Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp. (forexample, Fusarium gramineum, Fusarium venenatum), Physcomitrella patensand Neurospora crassa. Yeast, in particular, are currently preferredbecause yeast offers established genetics allowing for rapidtransformations, tested protein localization strategies, and facile geneknock-out techniques. Suitable vectors have expression controlsequences, such as promoters, including 3-phosphoglycerate kinase orother glycolytic enzymes, and an origin of replication, terminationsequences, and the like as desired.

Various yeasts, such as K. lactis, Pichia pastoris, Pichia methanolica,and Hansenula polymorpha are currently preferred for cell culturebecause they are able to grow to high cell densities and secrete largequantities of recombinant protein. Likewise, filamentous fungi, such asAspergillus niger, Fusarium sp, Neurospora crass, and others can be usedto produce recombinant proteins at an industrial scale.

Lower eukaryotes, in particular filamentous fungi and yeast, can begenetically modified so that they express proteins or glycoproteins inwhich the glycosylation pattern is human-like or humanized. This can beachieved by eliminating selected endogenous glycosylation enzymes and/orsupplying exogenous enzymes as described by Gerngross et al. in U.S.Pat. No. 7,029,872, and U.S. Published Patent Application Nos.20040018590, 20050170452, 20050260729, 20040230042, 20050208617,20040171826, 20050208617, 20060160179, 20060040353, and 20060211085.Thus, a host cell can additionally or alternatively be engineered toexpress one or more enzymes or enzyme activities, which enable theproduction of particular N-glycan structures at a high yield. Such anenzyme can be targeted to a host subcellular organelle in which theenzyme will have optimal activity, for example, by means of signalpeptide not normally associated with the enzyme. Host cells can also bemodified to express a sugar nucleotide transporter and/or a nucleotidediphosphatase enzyme. The transporter and diphosphatase improve theefficiency of engineered glycosylation steps, by providing theappropriate substrates for the glycosylation enzymes in the appropriatecompartments, reducing competitive product inhibition, and promoting theremoval of nucleoside diphosphates. See, for example, Gerngross et al.in U.S. Published Patent Application No. 20040018590 and Hamilton, 2003,Science 301: 1244-46 and the aforementioned U.S. patent and patentapplications.

By way of example, a host cell (for example, yeast or fungal) can beselected or engineered to be depleted in 1,6-mannosyl transferaseactivities, which would otherwise add mannose residues onto the N-glycanof a glycoprotein, and to further include a nucleic acid for ectopicexpression of an α-1,2 mannosidase activity, which enables production ofrecombinant glycoproteins having greater than 30 mole percentMan₅GlcNAc₂ N-glycans. When a glycoprotein is produced in the host cellsaccording to the method described herein, the host cells will produce aglycoprotein having predominantly a Man₅GlcNAc₂ N-glycan structure andreduced O-glycosylation compared to the glycoprotein produced in thecell otherwise. In a further aspect, the host cell is engineered tofurther include a nucleic acid for ectopic expression of GlcNActransferase I activity, which enables production of glycoproteins havingpredominantly GlcNAcMan5GlcNAc2 N-glycans. When a glycoprotein isproduced in the host cells according to the method described herein, thehost cells will produce a glycoprotein having predominantly aGlcNAcMan₅GlcNAc₂ N-glycan structure and reduced O-glycosylationcompared to the glycoprotein produced in the cell otherwise. In afurther still aspect, the host cell is engineered to further include anucleic acid for ectopic expression of mannosidase II activity, whichenables production of glycoproteins having predominantlyGlcNAcMan₃GlcNAc₂ N-glycans. When a glycoprotein is produced in the hostcells according to the method described herein, the host cells willproduce a glycoprotein having predominantly a GlcNAcMan₃GlcNAc₂ N-glycanstructure and reduced O-glycosylation compared to the glycoproteinproduced in the cell otherwise. In a further still aspect, the host cellis engineered to further include a nucleic acid for ectopic expressionof GlcNAc transferase II activity, which enables production ofglycoproteins having predominantly GlcNAc₂Man₃GlcNAc₂ N-glycans. When aglycoprotein is produced in the host cells according to the methoddescribed herein, the host cells will produce a glycoprotein havingpredominantly a GlcNAc₂Man₃GlcNAc₂ N-glycan structure and reducedO-glycosylation compared to the glycoprotein produced in the cellotherwise. In further still aspects, the above host cells can be furtherengineered to produce particular hybrid or complex N-glycan orhuman-like N-glycan structures by further including one or more highereukaryote genes involved in N-linked glycosylation, in any combination,that encode for example, sialytransferase activities, class II and IIImannosidase activities, GlcNAc transferase II, III, IV, V, VI, IXactivity, and galactose transferase activity. It is currently preferablethat the cells further include one or more of nucleic acids encodingUDP-specific diphosphatase activity, GDP-specific diphosphataseactivity, and UDP-GlcNAc transporter activity.

Plants and plant cell cultures may be used for expression of proteinsand glycoproteins with reduced O-linked glycosylation as taught herein(See, for example, Larrick & Fry, 1991, Hum. Antibodies Hybridomas 2:172-89); Benvenuto et al., 1991, Plant Mol. Biol. 17: 865-74); Durin etal., 1990, Plant Mol. Biol. 15: 281-93); Hiatt et al., 1989, Nature 342:76-8). Preferable plant hosts include, for example, Arabidopsis,Nicotiana tabacum, Nicotiana rustica, and Solanum tuberosum.

Insect cell culture can also be used to produce proteins andglycoproteins proteins and glycoproteins with reduced O-linkedglycosylation, as taught herein for example, baculovirus-basedexpression systems (See, for example, Putlitz et al., 1990,Bio/Technology 8: 651-654).

Although not currently as economical to culture as lower eukaryotes andprokaryotes, mammalian tissue cell culture can also be used to expressand produce proteins and glycoproteins with reduced O-linkedglycosylation as taught herein (See Winnacker, From Genes to Clones (VCHPublishers, N.Y., 1987). Suitable hosts include CHO cell lines, variousCOS cell lines, HeLa cells, preferably myeloma cell lines or the like ortransformed B-cells or hybridomas. Expression vectors for these cellscan include expression control sequences, such as an origin ofreplication, a promoter, an enhancer (Queen et al., 1986I, mmunol. Rev.89:49-68), and necessary processing information sites, such as ribosomebinding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Expression control sequences arepromoters derived from immunoglobulin genes, SV40, Adenovirus, bovinePapilloma Virus, cytomegalovirus and the like. Generally, a selectablemarker, such as a neoR expression cassette, is included in theexpression vector.

The nucleic acid encoding the protein to be expressed can be transferredinto the host cell by conventional methods, which vary depending on thetype of cellular host. For example, calcium phosphate treatment,protoplast fusion, natural breeding, lipofection, biolistics,viral-based transduction, or electroporation can be used for cellularhosts. Tungsten particle ballistic transgenesis is preferred for plantcells and tissues. (See, generally, Maniatis et al., Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Press, 1982))

Once expressed, the proteins or glycoproteins having reduced O-linkedglycosylation can be purified according to standard procedures of theart, including ammonium sulfate precipitation, affinity columns, columnchromatography, gel electrophoresis and the like (See, generally,Scopes, R., Protein Purification (Springer-Verlag, N.Y., 1982)).Substantially pure glycoproteins of at least about 90 to 95% homogeneityare preferred, and 98 to 99% or more homogeneity most preferred, forpharmaceutical uses. Once purified, partially or to homogeneity asdesired, the proteins can then be used therapeutically (includingextracorporeally) or in developing and performing assay procedures,immunofluorescent stainings, and the like. (See, generally,Immunological Methods, Vols. I and II (Lefkovits and Pernis, eds.,Academic Press, NY, 1979 and 1981).

Therefore, further provided are glycoprotein compositions comprising apredominant species of N-glycan structure and having reduced O-linkedglycosylation compared to compositions of the glycoprotein which havebeen produced in host cells have not been incubated in the presence ofan inhibitor of Pmt-mediated O-linked glycosylation or anα-1,2-mannosidase capable of trimming more than one mannose residue froma glycans structure or both. In particular aspects, the glycoproteincomposition comprises a glycoprotein having a predominant N-glycanstructure selected from the group consisting of Man₅GlcNAc₂,Man₃GlcNAc₂, GlcNAcMan₅GlcNAc₂, GlcNAcMan₃GlcNAc₂, GlcNAc₂Man₃GlcNAc₂,GalGlcNAcMan₅GlcNAc₂, Gal(GlcNAc)₂ Man₅GlcNAc₂, (GalGlcNAc)₂Man₅GlcNAc₂,NANAGalGlcNAcMan₃GlcNAc₂, NANA₂Gal₂GlcNAcMan₃GlcNAc₂, andGalGlcNAcMan₃GlcNAc₂ glycoforms.

III. Pharmaceutical Compositions

Proteins and glycoproteins having reduced O-linked glycosylation can beincorporated into pharmaceutical compositions comprising theglycoprotein as an active therapeutic agent and a variety of otherpharmaceutically acceptable components (See, Remington's PharmaceuticalScience (15th ed., Mack Publishing Company, Easton, Pa., 1980). Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions can also include, depending onthe formulation desired, pharmaceutically-acceptable, non-toxic carriersor diluents, which are defined as vehicles commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological phosphate-buffered saline, Ringer's solutions, dextrosesolution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation can also include other carriers, adjuvants,or nontoxic, nontherapeutic, nonimmunogenic stabilizers, and the like.

Pharmaceutical compositions for parenteral administration are sterile,substantially isotonic, pyrogen-free and prepared in accordance with GMPof the FDA or similar body. Glycoproteins can be administered asinjectable dosages of a solution or suspension of the substance in aphysiologically acceptable diluent with a pharmaceutical carrier thatcan be a sterile liquid such as water oils, saline, glycerol, orethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents, surfactants, pH buffering substances and the likecan be present in compositions. Other components of pharmaceuticalcompositions are those of petroleum, animal, vegetable, or syntheticorigin, for example, peanut oil, soybean oil, and mineral oil. Ingeneral, glycols such as propylene glycol or polyethylene glycol arepreferred liquid carriers, particularly for injectable solutions.Glycoproteins can be administered in the form of a depot injection orimplant preparation which can be formulated in such a manner as topermit a sustained release of the active ingredient. Typically,compositions are prepared as injectables, either as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection can also be prepared. The preparationalso can be emulsified or encapsulated in liposomes or micro particlessuch as polylactide, polyglycolide, or copolymer for enhanced adjuvanteffect, as discussed above (See Langer, Science 249, 1527 (1990) andHanes, Advanced Drug Delivery Reviews 28, 97-119 (1997).

Unless otherwise defined herein, scientific and technical terms andphrases used in connection with the present invention shall have themeanings that are commonly understood by those of ordinary skill in theart. Further, unless otherwise required by context, singular terms shallinclude the plural and plural terms shall include the singular.Generally, nomenclatures used in connection with, and techniques ofbiochemistry, enzymology, molecular and cellular biology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, for example, Sambrook et al. Molecular Cloning: ALaboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989); Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates (1992, and Supplementsto 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Taylor andDrickamer, Introduction to Glycobiology, Oxford Univ. Press (2003);Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold,N.J.; Handbook of Biochemistry: Section A Proteins, Vol I, CRC Press(1976); Handbook of Biochemistry: Section A Proteins, Vol II, CRC Press(1976); Essentials of Glycobiology, Cold Spring Harbor Laboratory Press(1999).

The following examples are intended to promote a further understandingof the present invention.

EXAMPLE 1

This example provides method for preparing various Pmt inhibitors.Unless otherwise stipulated all materials were obtained fromSigma-Aldrich Chemical Co. (St. Louis, Mo.) and used as received. The ¹HNMR spectra of all intermediates and final products were in accord withpublished data.

Preparation of Pmti-1,(5-[[3,4-bis(phenylmethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticAcid), is as follows.

The procedure was adapted from Orchard et al in U.S. Pat. No. 7,105,554.A solution of rhodanine-3-acetic acid (1 g, 5.20 mmol, 1 eq.),3,4-dibenzyloxybenzaldehyde (2.04 g, 6.25 mmol, 1.2 eq.), and sodiumacetate (1.3 g, 15.6 mmol, 3 eq.) in acetic acid (30 mL) is heated toreflux, and stirred overnight. As the reaction mixture is cooled to roomtemperature, the product is precipitated and filtered and washed withacetic acid, then petroleum ether. The residue is dissolved in hot DMSO,filtered, and precipitated by addition of water. Upon cooling, theprecipitate is filtered and recrystallized from ethyl acetate andpetroleum ether to give a product which is suspended in water andfreeze-dried overnight in vacuo to give the final product as a fluffyyellow powder.

Preparation of Pmti-2, 2(5-[[3-(1-Phenylethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticAcid), is as follows.

This product is synthesized according to the directions of Orchard etal. in U.S. Pat. No. 7,105,554. A solution of rhodanine-3-acetic acid(375 mg, 1.96 mmol, 1 eq.),3-(1-phenylethoxy)-4-(2-phenylethoxy)benzaldehyde (680 mg, 1.96 mmol,1eq.) and ammonium acetate (453 mg, 3 eq.) is heated to 70° C. for tenminutes, then cooled to room temperature and diluted with ethyl acetate(100 mL). The organic solution is washed with 1M HCl (2×200 mL) andbrine (200 mL) then dried over sodium sulfate and evaporated. Theproduct is purified by liquid chromatography using a 10×2.5 cm glasscolumn packed with 35-75 μm C18 (Alltech Associates, Deerfield, Ill.).Gradient elution is employed. Buffer A is 0.1% acetic acid and buffer Bis 80% acetonitrile. The gradient is comprised of 20% B for threeminutes, increasing to 75% B over 40 minutes. The flow rate is 8 mL/min.Detection is at 280 nm. The appropriate fractions are pooled,concentrated, and freeze-dried in vacuo to give the product as a fluffyyellow powder.

Preparation of Pmti-3,(5-[[3-(1-Phenyl-2-hydroxy)ethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticAcid), (Orchard et al. in U.S. Pat. No. 7,105,554) is synthesized inthree steps as follows.

Step 1: Production of (+)-(S)-2-Acetoxy-1-bromo-1-phenylethane. ColdHBr-acetic acid (12.4 g, 52.2 mmol) is added dropwise to(−)-(R)-1-phenylethane-1,2-diol(2.4 g, 17.4 mmol) during about fiveminutes and the mixture stirred at room temperature for 40 minutes.Water (25 mL) is added and the solution is neutralized with sodiumcarbonate and extracted with ether (3×30 mL). The combined extracts aredried and evaporated to give (+)-(S)-2-acetoxy-1-bromo-1- phenylethane(3.93 g, 93%), d²⁵ 1.415 g/mL, [x]₀ ²⁴+93.5° (c 5.63 in CCl₄) 2.72 (5H,s), 4.98 (aH, dd, 6.7 and 7.0 Hz) and 5.56 (2H, d). This product is notdistilled. The isomeric homogeneity is established by comparison of thenmr spectrum (absence of PhCH*OAc resonance) with that of1,2-diacetoxy-1-phenylethane. (Note that racemic reagents aresubstituted for the optical isomers listed).

Step 2: Production of3-[(1-Phenyl-2-hydroxy)ethoxy]-4-(2-phenylethoxy)-benzaldehyde.(2-Acetoxy-1-bromoethyl)benzene (3.32 g, 13.67 mmol, 1.2 eq) (theproduct of Step 1), is added to a stirred solution of3-hydroxy-4-(2-phenylethoxy)-benzaldehyde (2.76 g, 11.39 mmol, 1 eq.)and cesium carbonate (2.97 g, 9.11 mmol, 0.8 eq.) inN,N-dimethylformamide (15 mL). The solution is stirred for 19 hours atroom temperature, then 21 hours at 80° C. The reaction is worked up bypartitioning between ethyl acetate and water (brine is added to helpbreak up the emulsion that formed). The organic layer is washed twicemore with water, brine, and then dried over sodium sulfate andevaporated to give a dark oil. The residue is purified by chromatographyon silica gel and elution with diethyl ether gives an orange oil. Thisoil is dissolved in methanol (100 ml) and to the solution is added anaqueous solution of sodium hydroxide (7 mL, 1M). After 30 minutes, themixture is evaporated (to remove the methanol) and the residuepartitioned between dichloromethane and water. The organic layer isdried over sodium sulfate and evaporated. The residue is purified bychromatographed on silica gel and elution with petroleum ether:diethylether (1:2) gives the product as a cream coloured powder.

Step 3: Production of Pmti-3. A solution of rhodanine-3-acetic acid (158mg, 0.828 mmol, 1 eq.),3-(1-phenyl-2-hydroxy)ethoxy)-4-(2-phenylethoxy)benzaldehyde (300 mg,0.828 mmol, 1 eq.) (the product of Step 2), and ammonium acetate (191mg, 3 eq) in toluene (10 mL) is heated to reflux for 3.5 hours, cooledto room temperature, and diluted with ethyl acetate (50 mL). The organicsolution is washed with 1M HCl (2×200 mL) and brine (200 mL) then driedover sodium sulfate and evaporated. After work-up, the residue ispurified by chromatography on silica gel. Elution with ethyl acetategives a yellow gum, which is recrystallized from diethyl ether andpetroleum ether to give the product as a yellow powder.

EXAMPLE 2

This example shows that Pichia pastoris transformed with an expressionvector encoding the Kringle 1-3 marker glycoprotein and treated with Pmtinhibitors produced a glycoprotein having reduced O-glycosylation.

Plasmid DNA encoding a His-tagged reporter glycoprotein consisting ofhuman plasminogen domains K1, K2, and K3 (Kringle 1-3 protein) under thecontrol of the Pichia pastoris alcohol oxidase 1 (AOX1) promoter wastransformed into wild-type Pichia pastoris to produce strain yJC53. TheKringle reporter protein consisting of domains K1, K2, K3, and K4 hasbeen discussed in Duman et al. Biotechnol. Appl. Biochem. (1998), v.28,p. 39-45 and only domain K3 in Choi et al., 2003, Proc. Natl. Acad. Sci.U.S.A. 100(9): 5022-5027. The amino acid sequence of the Kringle 1-3protein used in the Example isSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHSRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDGGGHHHHHHHHH (SEQ ID NO: 1). The Kringle 1-3 protein contains at least twopotential mammalian O-glycosylation sites that conform to the purportedconsensus sequence P at −1 and +3: the serine residue, which isO-glycosylated, is capitalized in the amino acid sequence “pppSsgp” andthe threonine residue, which is O-glycosylated, is capitalized in theamino acid sequence “lapTapp”. The O-glycosylation sites are underlinedin the above amino acid sequence. The potential mammalianO-glycosylation sites are located between the K1 and K domains and theK2 and K3 domains. However, as shown in Table 1, in yeast the proteinhas about 20 O-linked glycosylation sites. Thus, O-linked glycosylationcan be a significant disadvantage to producing proteins in yeast withoutinhibiting O-linked glycosylation as shown by the methods herein. AnN-glycosylation site resides in the K3 domain, which had been removed byreplacing the asparagine at position 208 of SEQ ID NO: 1 with a serine.Therefore, the only glycans on the Kringle 1-3 protein would be theresult of O-glycosylation.

Plasmid containing DNA encoding the Kringle 1-3 protein was preparedusing forward primer K1-3/UP 5′-CGGAA TTCTC AGAGT GCAAG ACTGG GAATAGAA-3′ (SEQ ID NO:2) and reverse primer K1-3/LP1 (Reverse primer,3Gly+2H is, paired with K1-3/UP) 5′-ATGAT GATGA CCACC ACCGT CCTGG ACCACAGGGG TTAG-3′ (SEQ ID NO:3) to produce a PCR product, which was then PCRamplified using reverse primer K1-3/LP2 (Reverse primer, 3Gly+9His+stopcodon, paired with K1-3/UP) 5′-TTAAT GATGA TGATG ATGAT GATGA TGATG ACCACCACC-3′ (SEQ ID NO:4). PCR conditions were as follows: after 1 cycle of95° C. for 2 minutes as a denaturation step, the PCR reaction wassubjected to 30 cycles of 95° C. for 30 seconds, 60° C. for 30 seconds,and 72° C. for 1 minute, and then 1 cycle of 72° C. for 10 minutes.After the PCR reaction and column purification of the PCR products,nucleotide A overhangs of the PCR products were generated using ExTaq (1cycle of 72° C. for 15 minutes). The resulting PCR products were usedfor the second PCR reaction as a PCR template where the primers, K1-3/UPand K1-3/LP2, were used to amplify wild-type Kringle 1-3+3Gly+9His,which was cloned into a pCR2.1 plasmid vector (Invitrogen) to producepBK105. The following PCR primers were then used to generate an Asn toSer mutation at position 208 in the Kringle 1-3 protein to produce aminoacid sequence NRTP from amino acid sequence SRTP: forward primer K3f(Asn to Ser) 5′-ACCCCTCACACACATTCTAGGACACCAGAAAACTTC-3′ (SEQ ID NO:5)and reverse primer K3r 5′-CTGTGCACTCCAGTGCTGACAGGTGTG-3′ (SEQ ID NO:6).The Asn to Ser mutation was then generated in pBK105 by the Inverse PCR.PCR conditions were as follows; after 1 cycle of 95° C. for 2 minutes asa denaturation step, the PCR reaction was subjected to 35 cycles of 95°C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 5 minutes, andthen 1 cycle of 72° C. for 10 minutes. The resulting PCR products wereligated to produce plasmid pBK118, which was sequenced to confirm themutation.

Plasmid pBK118 was digested with EcoRI and the DNA fragments were gelpurified and cloned into the EcoRI sites of pPICZaA (Invitrogen, LaJolla, Calif.) to produce pBK119 (Pichia expression plasmid). PlasmidpPICZaA contains an α-factor secretion signal that allows the efficientsecretion of most proteins from Pichia pastoris; 5′-AOX, a 942 bpfragment containing the AOX 1 promoter that allows methanol-inducibleand high-level expression in Pichia pastoris; and, the ZEOCIN resistancegene for positive selection in E. coli and Pichia pastoris. Plasmid pBK119 was linearized with PineI before transforming into Pichia pastorisstrains. Plasmid pBK was transformed into Pichia pastoris strain yJC53,a wild-type strain, and various PMT knockout strains.

PMT knockout yeast strains were created in Pichia pastoris following theprocedure outlined for Saccharomyces cerevisiae in Gentzsch and Tanner,EMBO J. 1996 Nov. 1; 15(21): 25752-5759). The Pichia pastoris PMT geneswere identified in the nucleotide sequence of the Pichia pastoris genomeobtained from Integrated Genomics, Chicago, Ill. by homology searchingusing the nucleotide sequences for the Saccharomyces cerevisiae PMTgenes. Deletion of Pichia pastoris PMT (PpPMT genes was as follows. ThePpPMT deletion alleles were generated by the PCR overlap method (See forexample, Davidson et al., 2004, Glycobiology 14:399-407; Ho et al.,1989, Gene 77:51-9; Horton et al., 1989, Gene 77:61-8). In the first PCRreaction, DNA comprising the nucleotide sequences for 5′ and 3′ flankingregions of the PMT genes and the NAT or HYG resistance markers(Goldstein and McCusker, 1999, Yeast 14:1541-1553; Goldstein et al.,1999, Yeast 15:507-110) were PCR amplified. The primers sequences forthe regions flanking the PMT genes were designed using the Pichiapastoris genome nucleotide sequence obtained from Integrated Genomics,Chicago, Ill. as a guide. Pichia pastoris genomic DNA was used as atemplate for the PpPMT flanking regions PCR amplification, while NAT andHYG fragments were PCR amplified using plasmids as described in(Goldstein and McCusker, 1999, ibid.; Goldstein et al., 1999, ibid.) astemplates. Then, in a second PCR reaction, all three first round PCRproducts were used as templates to generate an overlap product thatcontained all three fragments as a single linear allele. The final PCRproduct was then directly employed for transformation. Transformantswere selected on YPD medium containing 200 μg/mL of hygromycin or 100μg/mL of nourseothricin. In each case the proper integration of themutant allele was confirmed by PCR. The PMT knockout strains createdwere yJC51 (pmt3Δ,pmt5Δ,pmt6Δ,), yJC55 (pmt1Δ), yJC66 (pmt2Δ), and yJC65(pmt4Δ). The PMT knockout strains were each transformed with plasmidpBK119 encoding the above Kringle 1-3 protein.

Kringle 1-3 protein expression for the transformed yeast strains wascarried out at in shake flasks at 24° C. with buffered glycerol-complexmedium (BMGY) consisting of 1% yeast extract, 2% peptone, 100 mMpotassium phosphate buffer pH 6.0, 1.34% yeast nitrogen base, 4×10-5%biotin, and 1% glycerol. The induction medium for protein expression wasbuffered methanol-complex medium (BMMY) consisting of 1% methanolinstead of glycerol in BMGY. Pmt inhibitor Pmti-1, Pmti-2, or Pmti-3 inmethanol was added to the growth medium to a final concentration of 0.2μM, 2 μM, or 20 μM at the time the induction medium was added. Cellswere harvested and centrifuged at 2,000 rpm for five minutes. The Pmtinhibitors Pmti-1, Pmti-2, and Pmti-3 are essentially interchangeable,with small variations in ease of use. For example, in the cell cultureconditions described, the solubility of Pmti-3 is greater than that ofPmti-1 and Pmti-2 and, therefore, the most desirable of the three.

Seven μL of the supernatant from the yJC53 cultures treated with Pmti-1or yJC55 was separated by polyacrylamide gel electrophoresis (SDS-PAGE)according to Laemmli, U. K. (1970) Nature 227, 680-685 and thenelectroblotted onto nitrocellulose membranes (Schleicher & Schuell (nowWhatman, Inc., Florham Park, N.J.). Kringle 1-3 protein was detected onthe Western blots using an anti-His antibody (H-15) from Santa CruzBiotechnology Inc. (Santa Cruz, Calif.) and developed using theImmunoPure Metal Enhanced DAB Substrate Kit (Pierce Biotechnology,Rockford, Ill.). As shown in Lane 1 of the Western blot shown in FIG. 1,Kringle 1-3 protein from untreated Pichia pastoris runs as a smear dueto the presence of O-glycosylation. However, in contrast, Kringle 1-3protein from yJC53 (Pichia pastoris treated with 2 or 20 μM Pmti-1,lanes 2 and 3, respectively) exhibits a distinct band, due to lack ofO-glycosylation, similar to that of Kringle 1-3 protein expressed fromyJC55 (a pmt1Δ knockout mutant of Pichia pastoris) (lanes 4 and 5). FIG.1 further shows that Pmti-1 reduced O-glycosylation to a level similarto that observed in a strain lacking Pmt1.

To measure O-glycosylation reduction by the Pmt inhibitors, the Kringle1-3 protein was purified from the growth medium using nickel chelationchromatography (Choi et al., 2003, Proc. Natl. Acad. Sci. U.S.A. 100(9):5022-5027) and the O-glycans released from and separated from Kringle1-3 protein by alkaline elimination (beta-elimination) (Harvey, 1999Mass Spectrometry Reviews 18, 349-451). This process also reduces thenewly formed reducing terminus of the released O-glycan (eitheroligomannose or mannose) to mannitol. The mannitol group thus serves asa unique indicator of each O-glycan. 0.5 nmole or more of Kringle 1-3protein, contained within a volume of 100 μL PBS buffer, was requiredfor beta elimination. The sample was treated with 25 μL alkalineborohydride reagent and incubated at 50° C. for 16 hours. About 20 μLarabitol internal standard was added, followed by 10 μL glacial aceticacid. The sample was then centrifuged through a Millipore filtercontaining both SEPABEADS and AG 50W—X8 resin and washed with water. Thesamples, including wash, were transferred to plastic autosampler vialsand evaporated to dryness in a centrifugal evaporator. 150 μl 1%AcOH/MeOH was added to the samples and the samples evaporated to drynessin a centrifugal evaporator. This last step was repeated five moretimes. 200 μL of water was added and 100 μL of the sample was analyzedby high pH anion-exchange chromatography coupled with pulsedelectrochemical detection-Dionex HPLC (HPAEC-PAD). Average O-glycanoccupancy was determined based upon the amount of mannitol recovered.The results are summarized in Table 1, which shows that any one of thePmt chemical inhibitors reduced O-linked glycosylation of secretedKringle 1-3 protein in the Pichia pastoris strains that contained intactPMT1 and PMT2 genes to a level that was comparable to the level ofO-linked glycosylation seen for cells containing deletions of either thePMT1 or PMT2 gene. Table 1 also shows that while the protein has twopotential mammalian O-linked glycosylation sites, in yeast the proteinhas about 20O-linked glycosylation sites.

TABLE 1 Strain Relevant Genotype Treatment O-Glycan Occupancy¹ yJC53Wild-Type 0 20 2 μM Pmti-1 9 yJC51 pmtΔ3, Δ5, Δ6 0 17 2 μM Pmti-1 6 20μM Pmti-1 4 0.2 μM Pmti-2 3 2 μM Pmti-2 2 0.2 μM Pmti-3 6 2 μM Pmti-3 4yJC55 pmtΔ1 0 3 2 μM Pmti-1 2 20 μM Pmti-1 2 yJC66 pmtΔ2 0 4 2 μM Pmti-14 20 μM Pmti-1 4 yJC65 pmtΔ4 0 18 2 μM Pmti-1 7 20 μM Pmti-1 4 ¹averagenumber of O-linked mannose chains per protein.

EXAMPLE 3

In this example, yeast cells transformed with DNA encoding the T. reeseiα-mannosidase results in production of proteins with reducedO-glycosylation and that the O-glycosylation was further reduced whenthe cells were also incubated in the presence of a Pmt inhibitor.

The H+L chains of an anti-Her2 monoclonal antibody were expressed inPichia pastoris strains GS115 (WT) and GS115 that was geneticallyengineered to co-expressed T. reesei α-mannosidase (+Trman). GS115 isavailable from Invitrogen (Carlsbad, Calif.) and, with the exception ofa HIS4 mutation to enable his4 selection, has an essentially wild typephenotype. The H+L chains were expressed as two separate genes fromplasmid pJC284, which was derived from Invitrogen plasmid pAO815.

The H+L genes were generated using anti-Her2 antibody sequences obtainedfrom GenBank. The GenBank accession number for the L chain is 1N8Z_A andthe GenBank accession number for the H chain variable region plus CH1domain is 1N8Z_B. The GenBank accession number for the H chain Fc regionis BC092518. Both the H and L chain DNA sequences were codon optimizedaccording to Pichia pastoris codon usage to enhance translation inPichia pastoris. Optimization of codons for use in Pichia sp. is wellknown in the art and has been described in, for example, Outchkourov etal., 2002,Protein Expr. Purif. 24:18-24; Sharp and Li, 1987, NucleicAcids Res. 15:1281-95; Woo J H, Liu et al., 2002, Protein Expression andPurification 25:270-282, and, Nakamura, et al., 2000, Nucleic Acids Res.28:292. Constant regions of the H chain (human IgG1) and L chain (humanKappa) were synthesized by GeneArt Inc., Regensburg, Germany. Variableregions were made in-house using oligonucleotides purchased from IDTInc. (Coralville, Iowa) in an overlapping PCR method. Full length H andL chains were assembled by overlapping PCR, and resulting H and L chainswere cloned into pCR2.1 TOPO vector (Invitrogen, La Jolla, Calif.) togenerate pDX344 and pDX349, respectively. H+L chains from pDX344 andpDX349 were combined with GAPDH promoters and AOX1 terminator sequencesin vector pDX580 (backbone derived from Invitrogen vector pGAPZA).Finally, the H+L chain expression cassettes were subcloned from pDX580into vector pJC284. The nucleotide sequence of the codon-optimized DNAencoding the light chain is shown in SEQ ID NO:7 and the nucleotidesequence of the codon-optimized DNA encoding the heavy chain is shown inSEQ ID NO:8. Plasmid pJC284 has GAPDH promoters for expressing the H+Lgenes and an intact his4 gene for selection of transformants in strainGS115 and GS115(+Trman). Yeast strains GS115 and GS115(+Trman) weretransformed with pJC285 and transformants with the plasmid integratedinto the genome at the his4 locus were isolated to produce strains thatproduced the anti-Her2 antibody.

Construction of strain GS115(+Trman) was as follows. The Trichodermareesei α-1,2-mannosidase was expressed from an expression cassette inplasmid pJC285. Plasmid pJC285 was derived from Invitrogen vectorpGAPZA, which has the Zeocin resistant gene as the selectable marker,and contains an expression cassette comprising DNA encoding the T.reesei α-1,2-mannosidase catalytic domain (SEQ ID NO:9) with the first84 base pairs encoding its signal sequence replaced with a DNA encodingthe Saccharomyces cerevisiea αMAT pre signal sequence (SEQ ID NO:10),which encodes just the ER-targeting amino acids, operably linked to DNAcomprising the Pichia pastoris GAPH promoter (SEQ ID NO: 11) at the 5′end and DNA comprising the Pichia pastoris AOX1 transcriptiontermination sequence (SEQ ID NO: 12) at the 3′ end. The nucleotidesequence of the complete expression cassette is set forth in SEQ ID NO:13. Yeast strain GS115 was transformed with pJC285 and transformantswith the plasmid integrated into the genome at the GAPDH locus wereisolated to produce strain GS115(+Trman).

Duplicate cultures of the strains were cultured in 200 mL of buffereddextrose-complex medium (BMDY) consisting of 1% yeast extract, 2%peptone, 100 mM potassium phosphate pH 6.0, 1.34% Yeast Nitrogen Base,0.00004% biotin, 2% dextrose, and with or without Pmti-2 at 0.3 or 0.03μM. Following 72 hours of growth, culture supernatants were collectedand centrifuged to remove yeast cells. Antibody in the remainingsupernatant fraction (about 200 mL) was purified over a Protein A columnand subjected to O-glycan analysis as described in Example 2. Inaddition to the mannitol assay, the average length of O-linked mannosechains was determined by chromatographic analysis without hydrolysis.The results are summarized in Table 2.

There are 14 yeast O-glycan sites on the antibody. When the antibody wasproduced in the wild-type GS115 strain, all 14 O-glycan sites haveglycan structures with 8% of the sites having just one mannose, 39%having a two mannose chain, 43% having a three mannose chain, and 9%having a four mannose chain (See Table 2). However, when the antibodywas produced in wild-type cells treated with the chemical inhibitorPmti-2, only two of the 14 O-glycan sites were occupied and for 76% ofthe two sites, the mannose chain had only one mannose residue. Neitherof the two sites had a mannose chain with three or four mannoseresidues. It should be noted that the analysis did not determine whichtwo of the 14 sites were occupied. Either any combination of twoO-glycan sites per antibody molecule were occupied orparticular-O-glycan sites are preferentially occupied. In the lattercase, to provide antibodies (or other proteins) completely devoid ofO-glycans, the amino acid sequences comprising the preferred O-glycansites can be modified to amino acid sequences that eliminates O-linkedglycosylation at the sites.

Table 2 further shows that when the antibody was produced in cells thatincluded DNA encoding the Tricoderma reesei α-1,2-mannosidase (strainGS115(+Trman)), only four of the 14 O-glycan sites were occupied and for95% of the four sites, the mannose chain had only one mannose residue.None of the four sites had a mannose chain with three or four mannoseresidues. If particular sites are preferentially O-glycosylated, toprovide antibodies (or other proteins) completely devoid of O-glycans,the amino acid sequences comprising the preferred O-glycan sites can bemodified to amino acid sequences that eliminates O-glycosylation at thesites.

Finally, Table 2 shows that when the antibody was produced in cells thatincluded DNA encoding the Tricoderma reesei α-1,2-mannosidase and in thepresence of Pmti-2, only one of the 14 O-glycan sites was occupied andfor 91% of the sites, the mannose chain had only one mannose residue. Nomannose chain had three or four mannose residues. If only one or only afew sites are preferentially O-glycosylated, to provide antibodies (orother proteins) completely devoid of O-glycans, the amino acid sequencescomprising the preferred O-glycan sites can be modified to amino acidsequences that eliminate O-glycosylation at the sites

Table 2 further shows that 0.3 uM of Pmti inhibitor is sufficient toreduced the occupancy by about 86% and the chain length for 76% of themolecules to one mannose while allowing the culture to grow. Includingthe Tricoderma reesei α-1,2-mannosidase allowed the amount of Pmtiinhibitor to be reduced by 10 fold and the occupancy reduced to 93% andthe chain length for 87% of the molecules to one mannose. These resultsshow that using an amount of Pmti inhibitor that does not kill the cellsis sufficient to produce glycoproteins having reduced O-linkedglycosylation. These results further show that the Pmti inhibitor andthe α-mannosidase appear to synergistically reduce the amount ofO-linked glycosylation.

TABLE 2 Strain Occupancy Man1 Man2 Man3 Man4 GS115 14 8 39 43 9 GS115 +0.3 μM Pmti-2 2 76 24 0 0 GS115(+Trman) 4 95 5 0 0 GS115(+Trman) + 0.3μM 1 91 9 0 0 Pmti-2 GS115(+Trman) + 0.03 μM 1 87 13 0 0 Pmti-2

Seven μL of the supernatant for each of the above were reduced andsubjected to SDS-PAGE and Western blotting using an HRP-conjugatedanti-human IgG (H&L) to detect H and L chains. The results are shown inFIG. 2. The hyper O-glycosylated H chain is the slowest migrating bandvisible in the first pair of lanes in FIG. 2. FIG. 2 shows that there isa decrease in the amount of O-glycosylated heavy chain when the antibodywas coexpressed with Tricoderma reesei α-1,2-mannosidase or the cellsexpressing the antibody was incubated in the presence of the Pmti-2inhibitor, or when the antibody was coexpressed with Tricoderma reeseiα-1,2-mannosidase and the cells expressing both proteins was incubatedin the presence of the Pmti-2 inhibitor.

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the claims attached herein.

1. A method of producing a protein having reduced O-linked glycosylationcomprising: (a) providing a nucleic acid encoding a protein operablylinked to an inducible promoter; (b) introducing the nucleic acid into ayeast host cell or filamentous fungal host cell to provide a culture ofthe host cell; (c) growing the culture for a time sufficient to providea multiplicity of the host cells having the nucleic acid beforecontacting the culture with one or more inhibitors of Pmt-mediatedO-linked glycosylation; (d) contacting the culture with an inducer ofthe promoter to induce expression of the protein at a time before or atthe same time as contacting the culture with the one or more inhibitorsof Pmt-mediated O-linked glycosylation; and (e) isolating theglycoprotein produced by the host cell in the presence of the one ormore inhibitors to produce the protein having reduced O-linkedglycosylation.
 2. The method of claim 1 wherein the one or moreinhibitors is a benzylidene thiazolidinedione.
 3. The method of claim 2wherein the one or more inhibitors are selected from the groupconsisting of:5-[[3,4-bis(phenylmethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticAcid;5-[[3-(1-Phenylethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticAcid; and5-[[3-(1-Penyl-2-hydroxy)ethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticAcid.
 4. The method of claim 1, wherein the host cell [is a yeast orfilamentous fungal cell that] has been genetically modified to produceglycoproteins with a predominant N-glycan glycoform.
 5. A method ofproducing a protein having reduced O-linked glycosylation comprising:(a) providing a nucleic acid encoding a protein operably linked to aninducible promoter; (b) introducing the nucleic acid into a yeast hostcell or filamentous fungal host cell and growing the host cellcontaining the nucleic acid to produce a culture of the host cell for atime sufficient to provide a multiplicity of the host cells having thenucleic acid before contacting the culture with one or more inhibitorsof Pmt-mediated O-linked glycosylation; (c) contacting the culture with[with one or both of]: (i) one or more inhibitors of Pmt-mediatedO-linked glycosylation and optionally with (ii) one or more [one ormore] of α-1,2-mannosidase enzymes; and (d) isolating the glycoproteinproduced by the host cell in the presence of the one or more inhibitorsand the one or more [one or more] optional α-1,2-mannosidase enzymes toproduce the protein having reduced O-linked glycosylation.
 6. The methodof claim 5 wherein the culture is grown in the presence of the one ormore inhibitors of Pmt-mediated O-linked glycosylation.
 7. The method ofclaim 5 wherein a second nucleic acid encoding the one or moreα-1,2-mannosidase enzymes is provided and [introducing] the secondnucleic acid is introduced into the host cell.
 8. The method of claim 5wherein the one or more inhibitors is a benzylidene thiazolidinedione.9. The method of claim 5 wherein the one or more inhibitors are selectedfrom the group consisting of:5-[[3,4-bis(phenylmethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticAcid;5-[[3-(1-Phenylethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticAcid; and5-[[3-(1-Phenyl-2-hydroxy)ethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticAcid.
 10. The method of claim 5 wherein the α-1,2-mannosidase is fromTrichoderma reesei, Saccharomyces sp., or Aspergillus sp.
 11. The methodof claim 5 wherein the α-1,2-mannosidase is from Trichoderma reesei. 12.The method of claim 5 wherein the host cell includes a second nucleicacid, which encodes the α-1,2-mannosidase, operably linked to aninducible promoter.
 13. The method of claim 5, wherein the host cell isselected from the group consisting of K. lactis, Pichia pastoris, Pichiamethanolica, and Hansenula.
 14. The method of claim 5, wherein the hostcell [is a yeast or filamentous fungal cell that] has been geneticallymodified to produce glycoproteins with a predominant N-glycan glycoform.